Human granulocyte colony stimulating factor has the main biological function of stimulating specific leucocytes, known as neutrophilic granulocytes or neutrophils, to accomplish growth and development thereof in vivo (Welte et al., PNAS, 82, 1526-1530, 1985: Souza et al Science, 232, 61-65, 1986). Such neutrophilic granulocytes function to protect a biological species against infection with microorganisms, when they are discharged to blood flow.
The amino acid sequence of human granulocyte colony stimulating factor has been reported by Nagata et al. (Nature, 319, 415-418, 1986). Human granulocyte colony stimulating factor is a protein capable of forming a complex with a receptor thereof in a ratio of 2:2 via the dimerization of the receptor (Horan et al., Biochemistry, 35, 4886-96, 1996).
Aritomi et al. have shown the X-ray structure of the BN-BC domain complex of human granulocyte colony stimulating factor with a receptor thereof (Nature, 401, 713-717, 1999). It is reported by Aritomi et al. that amino acids of human granulocyte colony stimulating factor, which exist in contact region or adjacent region thereof when the receptor is bound to human granulocyte colony stimulating factor, include G4, P5, A6, S7, S8, L9, P10, Q11, S12, L15, K16, E19, Q20, L108, D109, D112, T115, T116, Q119, E122, E123 and L124.
Some isoforms of human granulocyte colony stimulating factor, obtained by protein engineering technique have been disclosed (U.S. Pat. No. 5,581,476, U.S. Pat. No. 5,214,132, U.S. Pat. No. 5,362,853, U.S. Pat. No. 4,904,584). Ridehall-Olsen et al., have reported that Lys40, Val48, Leu49 and Phe144, present in the amino acid sequence of human granulocyte colony stimulating factor, participate in the bonding with a receptor of human granulocyte colony stimulating factor. Additionally, Rayton et al. have reported that Glu46, Leu49 and Phe144 are in contact with an immunoglobulin-like domain (Ig-like domain) of a receptor of human granulocyte colony stimulating factor, while Lys40 and Val49 are away from the domain. Thus, it is not certain that Lys40 and Val49 are in contact with the receptor (J. biol. Chem., 2001, 276, 36779-36787). Further, Rayton et al., have demonstrated that Glu19 of human granulocyte colony stimulating factor interacts with Arg288 of a receptor thereof, thereby serving to transfer the signals of human granulocyte colony stimulating factor (J. Biol. Chem., 1999, 274, 17445-17451).
It has been suggested to introduce at least one additional sugar chain artificially into the human granulocyte colony stimulating factor protein by a genetic engineering method (U.S. Pat. No. 5,218,092). Such introduction of the sugar chain is performed by using a method of exchanging, deleting and adding amino acids in the amino acid sequence of a polypeptide.
Additionally, many studies including bonding of polyethylene to human granulocyte colony stimulating factor have been reported (Satake-Ishikawa et al. Cell Structure and Function, 17, 157-160, 1992; U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,824,784, WO96/11953, WO95/21629, and WO94/20069).
In vivo removal of a polypeptide or a polymer thereof is made by removal (or clearance) and by receptor mediated degradation of protein in the kidney, spleen or liver. Such removal is accomplished by the action of a protease specific to a substrate or not. In general, in vivo protein removal depends on the size of protein (such a size that glomerular filtration can be prevented), the charge of a protein molecule, attachment of a sugar chain, protein receptor on the cell surface, or the like.
Particularly, protein removal in the kidney depends on the physical properties of a protein or a polymer thereof, such as size (molecular diameter), symmetry, shape/rigidity or charge, or the like.
Receptor mediated degradation of a protein is made when the protein loses its function upon the bonding with a receptor. At the initial time, leucocytes are insufficient, and bonding with a receptor on the surface of primordial hematopoietic stem cells results in differentiation and growth into leucocytes. However, once the number of leucocytes increases to reach a certain level, human granulocyte colony stimulating factor is removed by the receptor present on the surface of leucocytes in order to prevent excessive differentiation and growth caused by human granulocyte colony stimulating factor. The ratio of the primordial hematopoietic stem cells to the receptors of human granulocyte colony stimulating factor present on the surface of leucocytes is approximately 1:5.
Protein removal caused by the receptor present on the surface of leucocytes is executed by the introduction of a receptor-bound polypeptide into cells, and by lysosomal degradation of protein in the presence of proteases present in endosomes. Degradation of protein by bonding of a receptor to human granulocyte colony stimulating factor are described in detail by Saker and Roufenburger (Mol. Pharmacol., 2003, 63, 147-158) and Saker (Nature biotech. 2002, 20, 908-913).
To increase the half-life of a protein in blood, it is necessary to reduce protein removal in the kidney and degradation of the protein by bonding of a receptor. It is possible to reduce the protein removal in the kidney and to increase the half-life in vivo by bonding a polymer capable of increasing the apparent molecular size to the protein.
Moreover, adhesion of a polymer to a protein can interrupt proteases effectively and thus can prevent functions of non-specific proteases.
Among such polymers, polyethylene glycol (PEG) is one of the polymers widely used to prepare therapeutic protein products. Surface modification of a protein molecule caused by bonding of a protein drug to a synthetic polymer can increase the solubility of the drug to water or organic solvents. Accordingly, it is possible to increase the biocompatibility of the drug, to reduce the immunoreactivity, to improve the in vivo stability, and to retard clearance in the intestinal system, kidney, spleen or liver. Clearance of small protein molecules is made by filtering in the intestinal system or kidney. Hence, when a high-molecular weight PEG is bound to such small protein molecules, it is possible to prevent such clearance (Knauf, M. J. et al, J. Hiol. Chem. 263:15064, 1988). Introduction of a high-molecular weight polyethylene glycol (PEG) into a protein drug can increase the stability of the protein molecule in a solution. Additionally, it is possible to prevent adsorption of a non-specific protein by protecting the intrinsic surface characteristics of the protein effectively. In this regard, there has been an attempt to increase the in vivo half-life of a protein, to increase the solubility of a protein and to reduce the in vivo immunoreactivity by boding PEG to a biologically active peptide or protein. U.S. Pat. No. 4,179,337 reported the result of such attempts for the first time. Although bonding of PEG to a protein reduces many side effects due to the aforementioned advantages, the PEG-bound protein undesirably shows a significantly reduced in vivo activity because the active sites of the protein are blocked by PEG.
PEG, widely used up to date, is attached to the surface of protein by forming a covalent bond with at least one free lysine residue. Herein, if a site, which is present on the protein surface and is directly related to the activity of the protein, is bound to PEG, the activity of the protein decreases. Additionally, since the bonding between PEG and lysine residues occurs randomly, various kinds of PEG-bound protein conjugates are present in the form of a mixture, so that separation of a desired conjugate in a pure state becomes complicated and difficult. For example, EP 0401384 discloses a material and a method for producing polyethylene glycol-added G-CSF. EP0335423 discloses a modified polypeptide having an activity of human granulocyte colony stimulating factor. However, according to the prior art, the polyethylene glycol molecule cannot be bound to a predictable specific residue but bound to any reactive group present in the protein, such as a lysine residue, or the N-terminal in a non-specific manner, resulting in the formation of a non-homogeneous product. In order to bond a specific region of protein with PEG, U.S. Pat. No. 5,766,897 and WO00/42175 disclose a method of bonding human growth hormone with PEG, wherein PEG is allowed to react selectively with cysteine by using PEG-maleimide so as to prevent PEG from reacting with the active region of human growth hormone. It is required that a free cysteine residue that does not participate in disulfide bonding is present in human growth hormone to permit use of the above type of PEG. However, all of the four cysteine residues present in human growth hormone participate in disulfide bonding. Therefore, according to the prior art, pegylation is performed by using a human growth hormone derivative, into which a cysteine residue is artificially inserted. Additionally, U.S. Pat. No. 6,555,660 and U.S. Pat. No. 6,753,165 and US Laid-Open patent No. 2005/0058621 and KR2002-0079778 disclose mutation of cysteine at various positions in the amino acid sequence of human granulocyte colony stimulating factor.
Meanwhile, Bowen et al. have shown that molecular weight of polyethylene in polyethylene-modified human granulocyte colony stimulating factor is related to the lifetime of a drug. In an in vitro test, efficacy of a protein drug has an inverse relation with the molecular weight of polyethylene bound to the protein. However, in vivo activity increases as the molecular weight increases. It is thought that a polymer of a physiologically active protein shows a low affinity in receptor-mediated degradation, and thus shows an increased half life. Therefore, although such polymers of human granulocyte colony stimulating factor accomplish recovery of nutrophils in vivo, they show lower activity than the non-polymerized human granulocyte colony stimulating factor in terms of in vitro activity.
Recently, a modified human granulocyte colony stimulating factor that includes 20 kDa polyethylene glycol bound to the N-terminal has been developed and commercialized by Amgen, Inc. as Neulasta® (pegfilgrastim). The polyethylene glycol-polymerized human granulocyte colony stimulating factor has an increased half life, and thus can reduce administration frequency.
Particular examples of commercially available human granulocyte colony stimulating factor include E. coli-derived filgrastim (trade name: Gran and Neupogen), lenograstim (trade name: Neutroginand Granocyte) produced in animal cells, such as Chinese Hamster Ovary (CHO) cells, and E. coli-derived nartograstim (trade name: Neu-up), in which mutation occurs at the N-terminal regions in five amino acid sequences in order to increase the efficacy of human granulocyte colony stimulating factor protein.